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J. Biol. Chem., Vol. 262, Issue 34, 16364-16369, Dec, 1987
SO Sage and TJ Rink
We have investigated the sub-second kinetics of changes in cytosolic free
calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow
fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the
thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was
delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The
responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to
prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results
indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from
internal stores. The delays in onset of both responses are sufficient for
one or more biochemical steps to lie between ligand-receptor binding and
Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were
similar to those evoked by other agonists, but the response in the presence
of external Ca2+ was markedly shorter, occurring without measurable delay
at optimal ligand concentration. Analysis of this response showed some
delay in ADP- evoked influx at lower concentrations, but this delay was
markedly less than that observed with thrombin at doses giving the same
elevation in [Ca2+]i. These results suggest that ADP evokes influx using a
different transduction system, more closely coupled to the Ca2+ entry
system than that used by other agonists. Differences between thrombin- and
ADP- evoked influx were further demonstrated by the inhibitory actions of
cAMP, which reduced and substantially increased the delay in onset of
thrombin-evoked influx but did not measurably delay the influx evoked by an
optimal concentration of ADP.
The kinetics of changes in intracellular calcium concentration in fura- 2-loaded human platelets
Physiological Laboratory, University of Cambridge, United Kingdom.
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